This project has focused on discovering the molecular and biochemical mechanisms responsible for insulin resistance in human skeletal muscle in vivo. Our findings led us to focus on abnormalities in insulin signaling, specifically those involving IRS-1, and especially those defects that arise subsequent to lipid oversupply and inflammation. In the most recent funding period, we have discovered a number of novel phosphorylation sites in IRS-1 and showed how at least one of these sites functions to modulate IRS-1 action. In experiments designed to assess how lipid oversupply alters gene expression and insulin action in skeletal muscle, we discovered through a global gene expression analysis that lipid oversupply produces an inflammatory response in healthy skeletal muscle that results in increased synthesis of extracellular matrix proteins, MMPs, TIMPs, and connective tissue growth factor (CTGF), a mediator of TGF-p action. Preliminary results indicate that activation of the NF-KB pathway accompanied this response. This finding opens a new bridge between inflammation and muscle insulin resistance. Moreover, the inflammatory changes associated with insulin resistance are likely to be related to changes in IRS-1 serine/threonine phosphorylation, connecting these findings with the IRS-1 project. To mechanistically define the role of lipid- induced proinflammatory changes and to connect these changes with IRS-1 serine/threonine phosphorylation, we propose: 1) To develop a more complete serine/threonine phosphorylation map for IRS-1 and apply this map to characterize these sites in vitro and in vivo in humans in Aims 2-4. 2) To determine the time course of lipid-induced inflammatory events in skeletal muscle and circulating factors and connect these events with IRS-1 serine/threonine phosphorylation. 3) To use a local, femoral artery infusion of Liposyn to determine to what extent the lipid-induced inflammatory response and insulin resistance in skeletal muscle requires the influence of exogenous circulating factors. 4) To determine the extent to which the inflammatory response to lipids is present in naturally occurring insulin resistance. [unreadable] [unreadable] [unreadable]